RESUMO
The retropepsin (PR) of the Bovine leukemia virus (BLV) plays, as in other retroviruses, a crucial role in the transition from the non-infective viral particle to the infective virion by processing the polyprotein Gag. PR is expressed as an immature precursor associated with Gag, after an occasional -1 ribosomal frameshifting event. Self-hydrolysis of PR at specific N- and C-terminal sites releases the monomer that dimerizes giving rise to the active protease. We designed a strategy to express BLV PR in E. coli as a fusion protein with maltose binding protein, with a six-histidine tag at its N-terminal end, and bearing a tobacco etch virus protease hydrolysis site. This allowed us to obtain soluble and mature recombinant PR in relatively good yields, with exactly the same amino acid composition as the native protein. As PR presents relative promiscuity for the hydrolysis sites we designed four fluorogenic peptide substrates based on Förster resonance energy transfer (FRET) in order to characterize the activity of the recombinant enzyme. These substrates opened the way to perform kinetic studies, allowing us to characterize the dimer-monomer equilibrium. Furthermore, we obtained kinetic evidence for the existence of a conformational change that enables the interaction with the substrate. These results constitute a starting point for the elucidation of the kinetic properties of BLV-PR, and may be relevant not only to improve the chemical warfare against this virus but also to better understand other viral PRs.
Assuntos
Ácido Aspártico Proteases , Vírus da Leucemia Bovina , Dimerização , Escherichia coli/genética , Escherichia coli/metabolismo , Protease de HIV/metabolismo , Cinética , Vírus da Leucemia Bovina/genética , Vírus da Leucemia Bovina/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
Bovine leukemia virus (BLV) is a C-type retrovirus of cattle that causes huge economic losses with high infection rates in the majority of countries worldwide. To develop an anti-BLV vaccine, we constructed a peptide conjugate using the envelope glycoprotein gp51-peptide epitope, a putative receptor-binding site. This highly antigenic peptide was covalently linked to a mutant bacteriophage carrier (mQß) using two different linker strategies, isothiocyanate (NCS) and dinitrophenyl adipate. Both constructs elicited higher anti-BLV peptide IgG titers than the corresponding conjugate with keyhole limpet hemocyanin protein carrier (gold standard) in mice with the NCS linker strategy requiring less sample processing. The mQß-gp51-peptide construct is the first BLV peptide-based vaccine candidate to generate durable immunity (>539 days), which recognized both native gp51 protein and BLV particles and significantly decreased fusion of a susceptible cell line exposed to infectious BLV. These results support the high translational and animal health potential of the vaccine construct.
Assuntos
Vírus da Leucemia Bovina , Animais , Anticorpos Neutralizantes , Bovinos , Epitopos , Vírus da Leucemia Bovina/metabolismo , Camundongos , Peptídeos , Proteínas do Envelope Viral/metabolismoRESUMO
The aim of this study was to assess the protective effect and potential mechanism of melatonin against bisphenol A (BPA)-induced apoptosis and oxidative damage in FLK-BLV cells. The results showed that BPA reduced cell viability in a dose- and time-dependent manner, caused cell shrinkage and induced oxidative stress and apoptosis in FLK-BLV cells, which were effectively reversed by melatonin. In addition, BPA caused autophagy flux impairment, which was confirmed by the increased of LC3-II and p62 levels, whereas melatonin treatment effectively reduced p62 levels under BPA treatment, and reversed apoptosis-related protein expression patterns caused by BPA. However, inhibition of autophagy by CQ partially abolished the protective effect of melatonin on apoptosis, suggesting that melatonin against BPA-induced oxidative injury and apoptosis by activating autophagy pathway. Moreover, we found that melatonin inhibited BPA-induced the activation of p38 MAPK, which was comparable to SB203580 pretreatment, and companied by the activation of autophagy and decreases of apoptosis when compared to BPA alone, indicating that melatonin protected against BPA-induced apoptosis partially through the p38 MAPK-autophagy pathway. In conclusion, these results suggest that melatonin may prevent BPA-induced FLK-BLV cell damage by inhibiting p38/MAPK signaling pathway and activating autophagy, and it could be a potential therapeutic compound in preventing BPA-induced cell damage.
Assuntos
Vírus da Leucemia Bovina , Sistema de Sinalização das MAP Quinases , Melatonina , Animais , Apoptose , Autofagia , Compostos Benzidrílicos , Interações Medicamentosas , Vírus da Leucemia Bovina/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melatonina/farmacologia , Melatonina/uso terapêutico , Estresse Oxidativo , Fenóis , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Bovine leukemia virus (BLV)-induced tumoral development is a multifactorial phenomenon that remains incompletely understood. Here, we highlight the critical role of the cellular CCCTC-binding factor (CTCF) both in the regulation of BLV transcriptional activities and in the deregulation of the three-dimensional (3D) chromatin architecture surrounding the BLV integration site. We demonstrated the in vivo recruitment of CTCF to three conserved CTCF binding motifs along the provirus. Next, we showed that CTCF localized to regions of transitions in the histone modifications profile along the BLV genome and that it is implicated in the repression of the 5'Long Terminal Repeat (LTR) promoter activity, thereby contributing to viral latency, while favoring the 3'LTR promoter activity. Finally, we demonstrated that BLV integration deregulated the host cellular 3D chromatin organization through the formation of viral/host chromatin loops. Altogether, our results highlight CTCF as a new critical effector of BLV transcriptional regulation and BLV-induced physiopathology.
Assuntos
Vírus da Leucemia Bovina , Latência Viral , Fator de Ligação a CCCTC/metabolismo , Cromatina , Vírus da Leucemia Bovina/genética , Vírus da Leucemia Bovina/metabolismo , Regiões Promotoras Genéticas , Sequências Repetidas Terminais/genéticaRESUMO
Viruses have developed different strategies to escape from immune response. Among these, viral non-coding RNAs are invisible to the immune system and may affect the fate of the host cell. Bovine leukemia virus (BLV) encodes both short (miRNAs) and long (antisense AS1 and AS2) non-coding RNAs. To elucidate the mechanisms associated with BLV non-coding RNAs, we performed phenotypic and transcriptomic analyzes in a reverse genetics system. RNA sequencing of B-lymphocytes revealed that cell proliferation is the most significant mechanism associated with ablation of the viral non-coding RNAs. To assess the biological relevance of this observation, we determined the cell kinetic parameters in vivo using intravenous injection of BrdU and CFSE. Fitting the data to a mathematical model provided the rates of cell proliferation and death. Our data show that deletion of miRNAs correlates with reduced proliferation of the infected cell and lack of pathogenesis.
Assuntos
Linfócitos B , Transformação Celular Viral , Vírus da Leucemia Bovina , MicroRNAs , RNA Antissenso , RNA Viral , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos B/virologia , Bovinos , Proliferação de Células , Vírus da Leucemia Bovina/genética , Vírus da Leucemia Bovina/metabolismo , Vírus da Leucemia Bovina/patogenicidade , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle, which is closely related to human T-cell leukemia viruses. BLV has spread worldwide and causes a serious problem for the cattle industry. The cellular receptor specifically binds with viral envelope glycoprotein (Env), and this attachment mediates cell fusion to lead virus entry. BLV Env reportedly binds to cationic amino acid transporter 1 (CAT1)/solute carrier family 7 member 1 (SLC7A1), but whether the CAT1/SLC7A1 is an actual receptor for BLV remains unknown. Here, we showed that CAT1 functioned as an infection receptor, interacting with BLV particles. Cells expressing undetectable CAT1 levels were resistant to BLV infection but became highly susceptible upon CAT1 overexpression. CAT1 exhibited specific binding to BLV particles on the cell surface and colocalized with the Env in endomembrane compartments and membrane. Knockdown of CAT1 in permissive cells significantly reduced binding to BLV particles and BLV infection. Expression of CAT1 from various species demonstrated no species specificity for BLV infection, implicating CAT1 as a functional BLV receptor responsible for its broad host range. These findings provide insights for BLV infection and for developing new strategies for treating BLV and preventing its spread.-Bai, L., Sato, H., Kubo, Y., Wada, S., Aida, Y. CAT1/SLC7A1 acts as a cellular receptor for bovine leukemia virus infection.
Assuntos
Transportador 1 de Aminoácidos Catiônicos/metabolismo , Leucose Enzoótica Bovina/metabolismo , Vírus da Leucemia Bovina/metabolismo , Animais , Células CHO , Células COS , Transportador 1 de Aminoácidos Catiônicos/genética , Gatos , Bovinos , Chlorocebus aethiops , Cricetinae , Cricetulus , Leucose Enzoótica Bovina/virologia , Células HEK293 , Células HeLa , Humanos , Vírus da Leucemia Bovina/patogenicidade , Ligação Proteica , Ovinos , Suínos , Proteínas do Envelope Viral/metabolismoRESUMO
The envelope glycoprotein 51 (gp51) is essential for bovine leukaemia virus (BLV) entry to bovine B-lymphocytes. Although the bovine adaptor protein 3 complex subunit delta-1 (boAP3D1) has been proposed as the potential receptor, the specific ligand-receptor interaction has not yet been completely defined and boAP3D1 receptor and gp51 3D structures have not been determined. This study was thus aimed at a functional annotation of boAP3D1 cellular adaptor protein and BLV gp51 and, proposing a reliable model for gp51-AP3D1 interaction using bioinformatics tools. The boAP3D1 receptor interaction patterns were calculated based on models of boAP3D1 receptor and gp51 complexes' 3D structures, which were constructed using homology techniques and data-driven docking strategy. The results showed that the participation of 6 key amino acids (aa) on gp51 (Asn170, Trp127, His115, Ala97, Ser98 and Glu128) and 4 aa on AP3D1 (Lys925, Asp807, Asp695 and Arg800) was highly probable in the interaction between gp51 and BLVR domains. Three gp51 recombinant peptides were expressed and purified to validate these results: the complete domain (rgp51), the N-terminal portion (rNgp51) and the C-terminal fragment (rCgp51); and binding assays to Madin-Darby bovine kidney (MDBK) cells were then carried out with each recombinant. It was found that rNgp51 preferentially bound to MDBK cells, suggesting this domain's functional role during invasion. The rNgp51-MDBK cell interaction was sensitive to trypsin (98% reduction) and chymotrypsin treatment (80% reduction). These results highlighted that the N-terminal portion of gp51 interacted in vitro with the AP3D1 receptor and provides a plausible in silico interaction model.
Assuntos
Subunidades delta do Complexo de Proteínas Adaptadoras/metabolismo , Simulação por Computador , Vírus da Leucemia Bovina/metabolismo , Proteínas do Envelope Viral/metabolismo , Subunidades delta do Complexo de Proteínas Adaptadoras/química , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Anotação de Sequência Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Termodinâmica , Proteínas do Envelope Viral/químicaRESUMO
Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis, which results in significant economic losses on many affected farms. BLV infects a wide range of animals as well as cell lines derived from various mammalian species and organs; however, studies show that only some cell lines support sustained production of viral progeny. The differences between cells that produce viral progeny and those that do not are unclear. The aim of this study was to identify the steps of BLV replication that are associated with the capacity of a cell to support a productive infection. Eleven cell lines derived from various species were categorized into two groups, those that produce BLV progeny and those that do not, and the efficiency of viral attachment was compared. In addition, viral entry and reverse transcription were compared for two BLV-producing cell lines and three non-producing cell lines. BLV attached to and entered all of the tested cells. However, synthesis of viral DNA was inhibited in all three non-virus-producing cell lines, suggesting that BLV production was blocked either prior to or at the stage of reverse transcription. These results increase our understanding of the BLV life cycle and should enable better control over the spread of BLV.
Assuntos
DNA Complementar/genética , Resistência à Doença/genética , Células Epiteliais/virologia , Vírus da Leucemia Bovina/genética , RNA Viral/genética , Replicação Viral/fisiologia , Animais , Gatos , Bovinos , Linhagem Celular , Quirópteros , Chlorocebus aethiops , DNA Complementar/biossíntese , DNA Complementar/imunologia , Cães , Células Epiteliais/classificação , Células Epiteliais/imunologia , Humanos , Vírus da Leucemia Bovina/metabolismo , Camundongos , Especificidade de Órgãos , RNA Viral/metabolismo , Coelhos , Ratos , Transcrição Reversa , Especificidade da Espécie , Ligação Viral , Internalização do VírusRESUMO
Bovine leukemia virus (BLV) causes enzootic bovine leukosis and is closely related to the human T cell leukemia virus. Since BLV infection mostly occurs via cell-to-cell transmission, BLV infectivity is generally measured by culturing BLV-infected cells with reporter cells that form syncytia upon BLV infection. However, this method is time-consuming and requires skill. To visualize the infectivity of BLV, we developed a new assay called the luminescence syncytium induction assay (LuSIA) that is based on a new reporter cell line designated CC81-BLU3G. CC81-BLU3G is stably transfected with pBLU3-EGFP, which contains the BLV long terminal repeat U3 region linked to the enhanced-green fluorescence protein (EGFP) gene. CC81-BLU3G expresses the EGFP in response to BLV Tax expression specifically, and forms fluorescing syncytia when transfected with an infectious BLV plasmid or when cultured with BLV-infected cells. Compared to the conventional assay, LuSIA was more specific and detected cattle samples with low proviral loads. The fluorescing syncytia was easily detected by eye and automated scanning and LuSIA counts correlated strongly with the proviral load of infected cattle (R2 = 0.8942).
Assuntos
Bioensaio , Células Epiteliais/virologia , Células Gigantes/virologia , Vírus da Leucemia Bovina/genética , Medições Luminescentes/métodos , Provírus/genética , Animais , Células CHO , Gatos , Bovinos , Linhagem Celular Transformada , Cricetulus , Genes Reporter , Células Gigantes/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Vírus da Leucemia Bovina/crescimento & desenvolvimento , Vírus da Leucemia Bovina/metabolismo , Luminescência , Plasmídeos/química , Plasmídeos/metabolismo , Provírus/crescimento & desenvolvimento , Provírus/metabolismo , Ovinos , TransfecçãoRESUMO
Short hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, typical U6/H1 promoters encompass the majority of the â¼270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by >2-fold as compared to the typical U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting.
Assuntos
Marcação de Genes/métodos , Vírus da Leucemia Bovina/genética , MicroRNAs/genética , RNA Polimerase III/genética , RNA Interferente Pequeno/genética , RNA Viral/genética , Algoritmos , Pareamento de Bases , Sequência de Bases , Regulação da Expressão Gênica , Inativação Gênica , Genes Reporter , Vetores Genéticos , Células HEK293 , Humanos , Vírus da Leucemia Bovina/metabolismo , Luciferases/genética , Luciferases/metabolismo , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase III/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Análise de Sequência de RNA , Interface Usuário-ComputadorRESUMO
Programmed death-1 (PD-1), an immunoinhibitory receptor on T cells, is known to be involved in immune evasion through its binding to PD-ligand 1 (PD-L1) in many chronic diseases. We previously found that PD-L1 expression was upregulated in cattle infected with bovine leukemia virus (BLV) and that an antibody that blocked the PD-1/PD-L1 interaction reactivated T-cell function in vitro. Therefore, this study assessed its antivirus activities in vivo. First, we inoculated the anti-bovine PD-L1 rat monoclonal antibody 4G12 into a BLV-infected cow. However, this did not induce T-cell proliferation or reduction of BLV provirus loads during the test period, and only bound to circulating IgM+ B cells until one week post-inoculation. We hypothesized that this lack of in vivo effects was due to its lower stability in cattle and so established an anti-PD-L1 rat-bovine chimeric antibody (Boch4G12). Boch4G12 was able to bind specifically with bovine PD-L1, interrupt the PD-1/PD-L1 interaction, and activate the immune response in both healthy and BLV-infected cattle in vitro. Therefore, we experimentally infected a healthy calf with BLV and inoculated it intravenously with 1 mg/kg of Boch4G12 once it reached the aleukemic (AL) stage. Cultivation of peripheral blood mononuclear cells (PBMCs) isolated from the tested calf indicated that the proliferation of CD4+ T cells was increased by Boch4G12 inoculation, while BLV provirus loads were significantly reduced, clearly demonstrating that this treatment induced antivirus activities. Therefore, further studies using a large number of animals are required to support its efficacy for clinical application.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antivirais/uso terapêutico , Antígeno B7-H1/imunologia , Leucose Enzoótica Bovina/tratamento farmacológico , Vírus da Leucemia Bovina/metabolismo , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Reações Antígeno-Anticorpo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Bovinos , Células Cultivadas , Leucose Enzoótica Bovina/prevenção & controle , Leucose Enzoótica Bovina/virologia , Interferon gama , Vírus da Leucemia Bovina/fisiologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Carga ViralRESUMO
A two-month-old calf was diagnosed with leukosis on the basis of the clinical sign of enlarged, superficial lymph nodes. Serological and genetic tests for bovine leukemia virus (BLV) were performed because the calf was born from a cow infected with BLV. The serum had a weakly positive BLV antibody, and the BLV provirus was detected within neoplastic cells on performing polymerase chain reaction (PCR). Analysis of the BLV provirus integration site using inverse PCR revealed that the BLV integration site location was identical on all chromosomes in all tumor tissues examined. Thus, the tumor cells monoclonally proliferated following BLV infection. The present study shows that enzootic bovine leukosis can occur in a young animal, as in the two-month-old calf in our study.
Assuntos
Anticorpos Antivirais/biossíntese , DNA Viral/biossíntese , Leucose Enzoótica Bovina/virologia , Vírus da Leucemia Bovina/patogenicidade , Linfonodos/virologia , Animais , Bovinos , Proliferação de Células , Células Clonais , DNA Viral/genética , Leucose Enzoótica Bovina/diagnóstico , Leucose Enzoótica Bovina/patologia , Transmissão Vertical de Doenças Infecciosas , Vírus da Leucemia Bovina/genética , Vírus da Leucemia Bovina/metabolismo , Leucócitos/patologia , Leucócitos/virologia , Linfonodos/patologia , Masculino , Provírus/genética , Provírus/metabolismo , Integração ViralRESUMO
In a perspective of a comparative virology approach, characterization of the bovine leukemia virus (BLV) model may be helpful to better understand infection by the related human T-lymphotropic virus type 1 (HTLV-1). In this paper, we first provide detailed protocols to inoculate cloned BLV proviruses into sheep or cattle. We also describe methods to quantify apoptosis ex vivo and cell turnover in vivo.
Assuntos
Leucose Enzoótica Bovina/metabolismo , Vírus da Leucemia Bovina/metabolismo , Modelos Biológicos , Animais , Apoptose , Bovinos , Leucose Enzoótica Bovina/patologia , Vírus Linfotrópico T Tipo 1 Humano , Humanos , OvinosRESUMO
To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B' family of the regulatory subunits. B'-PP2A and HTLV-1 IN display nuclear co-localization, and the B' subunit stimulates concerted strand transfer activity of δ-retroviral INs in vitro. The protein-protein interaction interface maps to a patch of highly conserved residues on B', which when mutated render B' incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity.
Assuntos
Deltaretrovirus/metabolismo , Integrases/metabolismo , Proteína Fosfatase 2/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Bovinos , Linhagem Celular , Deltaretrovirus/enzimologia , Ativação Enzimática , Vírus Linfotrópico T Tipo 1 Humano/enzimologia , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Vírus da Leucemia Bovina/enzimologia , Vírus da Leucemia Bovina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteína Fosfatase 2/química , Subunidades Proteicas , Alinhamento de Sequência , Integração ViralRESUMO
UNLABELLED: Viruses have coevolved with their host to ensure efficient replication and transmission without inducing excessive pathogenicity that would indirectly impair their persistence. This is exemplified by the bovine leukemia virus (BLV) system in which lymphoproliferative disorders develop in ruminants after latency periods of several years. In principle, the equilibrium reached between the virus and its host could be disrupted by emergence of more pathogenic strains. Intriguingly but fortunately, such a hyperpathogenic BLV strain was never observed in the field or designed in vitro. In this study, we sought to understand the role of envelope N-linked glycosylation with the hypothesis that this posttranslational modification could either favor BLV infection by allowing viral entry or allow immune escape by using glycans as a shield. Using reverse genetics of an infectious molecular provirus, we identified a N-linked envelope glycosylation site (N230) that limits viral replication and pathogenicity. Indeed, mutation N230E unexpectedly leads to enhanced fusogenicity and protein stability. IMPORTANCE: Infection by retroviruses requires the interaction of the viral envelope protein (SU) with a membrane-associated receptor allowing fusion and release of the viral genomic RNA into the cell. We show that N-linked glycosylation of the bovine leukemia virus (BLV) SU protein is, as expected, essential for cell infection in vitro. Consistently, mutation of all glycosylation sites of a BLV provirus destroys infectivity in vivo. However, single mutations do not significantly modify replication in vivo. Instead, a particular mutation at SU codon 230 increases replication and accelerates pathogenesis. This unexpected observation has important consequences in terms of disease control and managing.
Assuntos
Vírus da Leucemia Bovina/genética , Vírus da Leucemia Bovina/patogenicidade , Proteínas do Envelope Viral/genética , Replicação Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Gatos , Fusão Celular , Chlorocebus aethiops , Glicosilação , Células HEK293 , Células HeLa , Humanos , Vírus da Leucemia Bovina/metabolismo , Fusão de Membrana/genética , Mutação , Estabilidade Proteica , Alinhamento de Sequência , Análise de Sequência de RNA , Ovinos , Proteínas do Envelope Viral/metabolismo , Carga ViralRESUMO
Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two other retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable.
Assuntos
Vírus da Leucemia Bovina/metabolismo , Chaperonas Moleculares/metabolismo , Ácidos Nucleicos/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Leucose Enzoótica Bovina/virologia , Infecções por HIV/virologia , HIV-1/química , HIV-1/metabolismo , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/química , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Vírus da Leucemia Bovina/química , Modelos Moleculares , Chaperonas Moleculares/química , Dados de Sequência Molecular , Ácidos Nucleicos/química , Proteínas do Nucleocapsídeo/química , Ligação ProteicaRESUMO
Exosomes are small membranous microvesicles (40-100 nm in diameter) and are extracellularly released from a wide variety of cells. Exosomes contain microRNA, mRNA, and cellular proteins, which are delivered into recipient cells via these exosomes, and play a role in intercellular communication. In bovine leukemia virus (BLV) infection of cattle, although it is thought to be a minor route of infection, BLV can be transmitted to calves via milk. Here, we investigated the association between exosomes and BLV in bovine milk. BLV structural proteins, gp51 (Env) and p24 (Gag), were detected in bovine milk exosomes from BLV-infected cattle by Western blot analysis. In cells inoculated with these milk exosomes, BLV DNA was not detected during three serial passages by nested PCR. Purification of exosomes from persistently BLV-infected cells was achieved by immuno-magnetic separation using an antibody against exosomes coupled to magnetic beads. Consistently, BLV gp51 and p24 proteins were detected in purified exosomes. Moreover, reverse transcriptase activity was observed in purified exosomes, meaning that exosomes also contain viral enzyme. However, BLV DNA was not detected in serially passaged cells after inoculation of purified exosomes, indicating that exosomes carrying BLV proteins appeared to be not infectious. These results suggest that BLV proteins are released with milk exosomes and could be transferred into recipient cells of calves via milk exosomes as an alternative route not requiring virus infection. Moreover it is also possible that bovine milk exosomes play a role in clearance of BLV proteins from infected cells.
Assuntos
Leucose Enzoótica Bovina/transmissão , Exossomos/metabolismo , Vírus da Leucemia Bovina/metabolismo , Leite/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Bovinos , Linhagem Celular , Ativação Enzimática , Exossomos/ultraestrutura , Vírus da Leucemia Bovina/genética , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle) or via clonal expansion of the infected cells (mitotic cycle). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone). We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1) initial integration inside genes or promoters and (2) host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in transcribed regions.
Assuntos
Leucose Enzoótica Bovina/metabolismo , Genoma , Vírus da Leucemia Bovina/metabolismo , Provírus/metabolismo , Transcrição Gênica , Integração Viral , Animais , Bovinos , Leucose Enzoótica Bovina/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Vírus da Leucemia Bovina/genética , Provírus/genéticaRESUMO
It is widely accepted that the majority of cancers result from multiple cellular events leading to malignancy after a prolonged period of clinical latency, and that the immune system plays a critical role in the control of cancer progression. Bovine leukemia virus (BLV) is an oncogenic member of the Retroviridae family. Complete genomic sequences of BLV strains isolated from peripheral blood mononuclear cells (PBMC) from cattle have been previously reported. However, a detailed characterization of the complete genome of BLV strains directly isolated from bovine tumors is much needed in order to contribute to the understanding of the mechanisms of leukemogenesis induced by BLV in cattle. In this study, we performed a molecular characterization of BLV complete genomes from bovine B-cell lymphosarcoma isolates. A nucleotide substitution was found in the glucocorticoid response element (GRE) site of the 5' long terminal repeat (5'LTR) of the BLV isolates. All amino acid substitutions in Tax previously found to be related to stimulate high transcriptional activity of 5'LTR were not found in these studies. Amino acid substitutions were found in the nucleocapsid, gp51 and G4 proteins. Premature stop-codons in R3 were observed. Few mutations or amino acid substitutions may be needed to allow BLV provirus to achieve silencing. Substitutions that favor suppression of viral expression in malignant B cells might be a strategy to circumvent effective immune attack.
Assuntos
Leucose Enzoótica Bovina/virologia , Genoma Viral , Vírus da Leucemia Bovina/genética , Linfoma de Células B/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Vírus da Leucemia Bovina/química , Vírus da Leucemia Bovina/metabolismo , Linfoma de Células B/virologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/veterinária , Alinhamento de Sequência/veterináriaRESUMO
Retroviral genome recognition is mediated by interactions between the nucleocapsid (NC) domain of the virally encoded Gag polyprotein and cognate RNA packaging elements that, for most retroviruses, appear to reside primarily within the 5'-untranslated region (5'-UTR) of the genome. Recent studies suggest that a major packaging determinant of bovine leukemia virus (BLV), a member of the human T-cell leukemia virus (HTLV)/BLV family and a non-primate animal model for HTLV-induced leukemogenesis, resides within the gag open reading frame. We have prepared and purified the recombinant BLV NC protein and conducted electrophoretic mobility shift and isothermal titration calorimetry studies with RNA fragments corresponding to these proposed packaging elements. The gag-derived RNAs did not exhibit significant affinity for NC, suggesting an alternate role in packaging. However, an 83-nucleotide fragment of the 5'-UTR that resides just upstream of the gag start codon binds NC stoichiometrically and with high affinity (K(d)=136±21 nM). These nucleotides were predicted to form tandem hairpin structures, and studies with smaller fragments indicate that the NC binding site resides exclusively within the distal hairpin (residues G369-U399, K(d)=67±8 nM at physiological ionic strength). Unlike all other structurally characterized retroviral NC binding RNAs, this fragment is not expected to contain exposed guanosines, suggesting that RNA binding may be mediated by a previously uncharacterized mechanism.